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Even though all living things have a lot in common, different organisms can have very different reactions to the same organic chemicals. That means it’s really important for organic chemists to be able to purify chemicals and separate the products we want from reactions, from the side products we don’t. In this episode of Crash Course Organic Chemistry, we’re heading into the lab to learn about one of the ways we can separate chemicals in a mixture: chromatography!

Episode Sources:
Mahdi, J. G. (2010). Medicinal potential of willow: A chemical perspective of aspirin discovery. Journal of Saudi Chemical Society, 14(3), 317-322.
Dempsey, D.A., Klessig, D.F. How does the multifaceted plant hormone salicylic acid combat disease in plants and are similar mechanisms utilized in humans?. BMC Biol 15, 23 (2017).
Still, W. C., Kahn, M., & Mitra, A. (1978). Rapid chromatographic technique for preparative separations with moderate resolution. The Journal of Organic Chemistry, 43(14), 2923-2925.
Pastre, J. C., & Duarte Correia, C. R. (2006). Efficient Heck arylations of cyclic and acyclic acrylate derivatives using arenediazonium tetrafluoroborates. A new synthesis of the antidepressant drug (±)-Paroxetine. Organic letters, 8(8), 1657-1660.

Series Sources:
Brown, W. H., Iverson, B. L., Ansyln, E. V., Foote, C., Organic Chemistry; 8th ed.; Cengage Learning, Boston, 2018.
Bruice, P. Y., Organic Chemistry, 7th ed.; Pearson Education, Inc., United States, 2014.
Clayden, J., Greeves, N., Warren., S., Organic Chemistry, 2nd ed.; Oxford University Press, New York, 2012.
Jones Jr., M.; Fleming, S. A., Organic Chemistry, 5th ed.; W. W. Norton & Company, New York, 2014.
Klein., D., Organic Chemistry; 1st ed.; John Wiley & Sons, United States, 2012.
Louden M., Organic Chemistry; 5th ed.; Roberts and Company Publishers, Colorado, 2009.
McMurry, J., Organic Chemistry, 9th ed.; Cengage Learning, Boston, 2016.
Smith, J. G., Organic chemistry; 6th ed.; McGraw-Hill Education, New York, 2020.
Wade., L. G., Organic Chemistry; 8th ed.; Pearson Education, Inc., United States, 2013.

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Hi! I’m Deboki Chakravarti and welcome  to Crash Course Organic Chemistry!

When you get down to it,  all life is kinda similar. From plants to fungi to humans, we share  many of the same biological organic reactions   that break down molecules for energy and  build the molecules that make you... you. But we are different from a mushroom or a tree.

And those differences allow some organisms to  produce molecules that may have wildly different effects in other organisms— sometimes  harmful, but sometimes beneficial. For example, willow leaves contain  salicylic acid that acts as a plant hormone,   and as far back as 4000 BCE the Assyrians used  this extract to treat joint pain in humans. Natural products like salicylic  acid can be medicines themselves,   or they can be inspiration  for more effective treatments.

Like in 1853, French chemist Charles Frédéric  Gerhardt synthesized a similar compound,   acetylsalicylic acid, which  we know today as aspirin. Gerhardt's aspirin wasn’t pure  acetylsalicylic acid, though,   which brings us to the topic of this episode. When we’re running reactions in a lab, we need to purify them to isolate the chemical we're trying to make from any other reactants or side products hanging around in the mixture.

And one method to separate  chemicals is chromatography! [Theme Music]. Many reactions produce mixtures, sometimes due to competing side reactions and sometimes because  the starting material didn’t completely react. For example, let’s look at a synthesis of  the antidepressant medication paroxetine,   also called Paxil.

We’re starting with an alcohol and  our goal is to turn it into an ether. Remember that hydroxide is a poor  leaving group, so in this synthesis,   we react the alcohol to form a mesylate. Now we have a great leaving group, and can use the Williamson etherification  reaction to make the ether.

Unfortunately, those reactions  aren’t super efficient,   and the ether is only 56% of our products. So before we move onto the  last step to make Paxil,   we’ll need to separate the ether we  want from side products that we don't. This is where chromatography comes in.

Chromatography is what we call a group  of different techniques that let us separate the components of a mixture,  usually to quantify or identify them. Chromatography always involves two key pieces to  help separate different molecules in a sample: there's a stationary phase or adsorbent,  and a mobile phase or eluant.   The inventor of chromatography, Russian-Italian  botanist Mikhail Tsvet, first used solid calcium carbonate as a stationary phase to  separate a sample of colored plant pigments. That’s how chromatography got its name!

We can do a simple kind of paper chromatography  using a piece of paper as our stationary phase,   to separate the component  pigments in washable marker ink. Paper is made up of cellulose, which is a  bunch of sugar molecules strung together. All of those hydroxyl groups hanging off  the sugars have dipoles, which means this paper is polar and interacts a lot  with molecules that are also polar.

We're not just tearing off a  piece of our notebook here. Notebook paper is often coated to prevent ink  from running, and we need the ink to spread out. Now, I’ll draw a little line on the bottom of our  chromatography paper in pencil to mark the origin,   which is where we put our  washable marker ink sample.

This ink is a mixture made up of several different  colored compounds that have different polarity. Then, we can stand the paper  up in a solvent chamber. Here, I just have a jar with our solvent,   or mobile phase: a mixture of  isopropyl alcohol and water.

We have to make sure the marker  ink spot is above the solvent,   or our sample will just diffuse into the  solution at the bottom of the chamber,   giving us a murky solution and  no separation on the paper. Oops. What we want to happen here is for a process called capillary action   to move the solvent up our chromatography paper.

We actually saw capillary action way back in  Episode 6 when we looked at candle wax, too! Polar compounds in the marker ink sample  spend a lot of time interacting with the polar stationary phase, and stick to the  cellulose molecules at the bottom of the paper. Less polar compounds don’t interact as much with the paper molecules and move up with the mobile phase more easily.

Different colored marker inks  have different compounds,   so let’s check out the beautiful color  separations from some paper chromatography! Like I mentioned, our solvent chamber  was filled with isopropyl alcohol   and water because that's safe and  easy to do in a Crash Course studio. But in organic chemistry labs, diethyl ether and  hexane is a common chromatography solvent system.

Also, because paper is porous, the colors  in the paper chromatography experiment were pretty spread out, which isn't that  useful for working with organic chemicals. So our stationary phase in the lab  is usually silica gel or alumina,   which has the consistency of very fine  sand and gives a better separation. A thin layer of the stationary phase  is adhered to a glass, aluminum,   or plastic-backed plate and we call the technique  thin layer chromatography, or TLC for short.

Now that we've got a new experimental  setup, let’s look at what happens to a sample of an organic compound when  we alter the ratios of solvents. We’ll start with an equal mixture of diethyl ether and hexane as our mobile  phase, a 1:1 solvent system. We can quantify the movement of a spot  of organic compound on the plate by calculating its retention factor or Rf –   a way to describe the eluding power of a solvent system,   or how much the solvent can move a compound  up a stationary phase during chromatography.

To calculate Rf, we measure the distance  traveled by the spot from the origin,   and divide it by the distance from  the solvent front to the origin,   which is how far we let the  solvent move up on the plate. We have to remove the plate from the  chamber before the solvent reaches the top,   otherwise the spot tends to spread out too much! Doing the math in this 1:1 solvent system,  our spot has a retention factor of 0.41.

Now let's try a different ratio by increasing the  hexane, the nonpolar solvent in our mobile phase,   for a 1:5 ratio of ether to hexane. After doing the chromatography experiment,  measuring, and calculating Rf, we only get 0.16! So we can say that the 1:5 solvent system has  less eluding power than the 1:1 solvent system.

The nonpolar solvent hexane is less effective at pulling polar organic   compounds away from the polar stationary phase and moving them up the TLC plate. However, if we switch the ratio and increase the  ether, the polar solvent in our mobile phase,   we'll get different results. With a 5:1 ratio of ether to hexane, we’ll expect polar organic compounds to interact to a greater extent with the more polar mobile phase, increasing the eluding power.

Let's do the math and, what do you know,  in this solvent system our Rf is 0.70. In the lab we're not just calculating Rf   values of spots of pure organic compounds in different solvents. We use chromatography to  separate reaction products,   like the ether from the side  products when making Paxil.

And that requires us to examine, and actually  physically separate, mixtures from a sample. We use TLC to monitor our reaction, and,   using a thin capillary tube, we put a spot of  our starting material in lane A on the left. We spot our reaction mixture, which usually contains multiple compounds that we want to separate from each other, in lane C on the right.

In the middle lane B, we spot both  on top of each other – a cospot. If all the lanes look like lane A after our  chromatography is done, we could have made   a completely pure product from the reaction  with an identical Rf to the starting material. However, it’s more likely that  something's gone wrong with the reaction.

So basically, TLC lets us preview  if our reaction actually worked,   or if we're stuck with the same  molecules that we started with! Lane B, the cospot, helps us make sure there really is a difference between our starting material and our reaction mixture,   and that we know our starting  material was used up in the reaction. For example, if lane B looks like it’s  missing the spot from lane A, but lanes B and C look the same, a starting material could've hitched a ride with the reaction solvent to move further up the plate and fool us into thinking it's a new compound when it's not.

But if lane B includes the same separations  as both lane A and lane C, we're good. It's really important to triple check  all this with these three lanes! Now, we’re not always working with colored  compounds in plants like Mikhail Tsvet,   or separating bright marker  ink with paper chromatography.

Often with TLC, our reaction mixtures are light  yellow or clear which can make them hard to see. Luckily, many organic compounds  have aromatic rings, double bonds,   or triple bonds, which absorb UV light. So we keep a UV light in the lab to shine on TLC plates and reveal our separated components, so we can circle our spots with a pencil.

If we're working with compounds that  don't show up under UV light, we use chromatography stains that can undergo chemical  reactions with the compounds on our plate. Conveniently, the effects of these  stains can be seen with the naked eye. For example, a potassium permanganate stain  reacts with compounds by oxidation, and makes the oxidized compound spots appear yellow, on a  pretty purplish background… kind of like our set!

TLC gives us a great preview of  how our compounds will separate,   but a tiny plate isn't practical to  purify grams of our reaction mixture. For that, we’ll need a bigger, badder  technique called flash chromatography. To set up our flash chromatography,   we actually need to play around with TLC to find a solvent system that gives the component we want to separate from the reaction mixture an Rf of about 0.2.

We also need to know the mass of the  unpurified sample we want to separate,   and from there we can pick an  appropriately sized flash column with a simple table – thanks to the work of chemists before us! So let's say that we've figured that out, and  now we’ll head into the Thought Bubble Lab! To start, we'll push a plug of cotton firmly  into the bottom of the chromatography column,   and cover the cotton with a  little sand for reinforcement.

We're gonna use a method called slurry  packing, where we mix the mobile phase solvent with silica gel as our stationary phase,  and pour that into the column through a funnel. Next, we’ll apply pressure to the  top of the column using an air hose,   until the solvent is level with the silica gel. Let’s add a little more sand  to keep the silica gel even,   and then we're ready to load  our sample into the column!

With a long Pasteur pipet, we add  our reaction mixture to the column. Then, we can fill the reservoir with  our mobile phase and run the column. We’ll apply pressure with an air hose again,   forcing the liquid through all the  silica gel, sand, and the cotton plug.

And we'll collect fractions of  the mobile phase in test tubes. We have to be careful not to let the solvent  move below the level of the silica gel in the column – that would mess up our carefully  balanced mixture of mobile and stationary phases. So we can add solvent to the top to  prevent the column from going dry!

The less-polar compounds move quickly through the silica gel in the flash column into the first few test tubes. After we've collected all the fractions, we can  check if our compound has eluded by doing TLC. The less-polar compounds will also move fastest  via capillary action upward on the TLC plate!

Lastly, we’ll combine the fractions that have the compound we want in a round bottom flask and concentrate using a rotary evaporator. A vacuum reduces the pressure inside the rotovap,   which lowers the boiling point of the solvents  to remove them easily from the sample. We spin the flask to create more surface area and encourage the solvent molecules to move into the vapor phase.

Hopefully we've isolated a good-sized,  pure sample of the compound we want! Thanks, Thought Bubble! With a purified sample, we can calculate a percent  yield to see how efficient our reaction was,   analyze our compound using spectroscopy, and then  move on to the next reaction in our synthesis.

Maybe now we have our purified ether and  can move onto the final step to make Paxil,   or we're doing any number of other reactions. Flash chromatography is pretty  common in organic chemistry! In this episode we learned that:.

Chromatography separates organic  compounds based on polarity. Thin layer chromatography tells  us how well our reaction went, and Flash chromatography lets us separate  larger quantities of compounds. In the next episode, we’ll talk about how we  can use proton NMR, one of the most important spectroscopy techniques to an organic chemist,  to see if we really made our desired product.

Until then, thanks for watching this  episode of Crash Course Organic Chemistry. If you want to help keep all  Crash Course free for everybody, forever, you can join our community on Patreon.